Method for identification, selection and analysis of tumour cells

ABSTRACT

The present invention relates to a method for the diagnosis of tumoural conditions and/or of the corresponding state of advance, wherein a specimen of an organic fluid taken from the patient and having a high probability of containing at least one circulating tumor cell (CTC) or a disseminated tumor cell (CTD) is enriched in a population of CTCs or CTDs. According to the invention, at least one type of CTCs or CTDs is isolated by selecting individually single cells in a microfluidic device so to constitute a diagnostic specimen having a purity of at least 90%. On the specimen thus obtained there is subsequently performed a molecular analysis such as to highlight a characteristic thereof suited to enabling diagnosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. national phase application of International ApplicationNo. PCT/IB2009/007316, filed Nov. 4, 2009, which claims the benefit ofItalian Patent Application No. TO2008A 000814, filed Nov. 4, 2008.

INCORPORATION BE REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application incorporates by reference in its entirety acomputer-readable nucleotide/amino acid sequence listing identified asfollows: One 607 byte ASCII (text) file name “46247_SeqListing.txt,”created Jan. 5, 2015.

IN THE SEQUENCE LISTING

Please introduce the computer-readable form of the Sequence Listingsubmitted herewith (filename: 46247_SeqListing.txt; Size: 607 bytes,Created: Jan. 5, 2015) as part of the application of theabove-identified application as filed.

FIELD OF THE ART

The present invention relates, in general, to diagnostic methods basedupon identification, isolation, and subsequent analysis of circulatingor disseminated rare cells. In particular, the invention relates todiagnostic methods that find application in the oncology sector, andthat hence involve identification and isolation of circulating tumourcells (CTCs) or disseminated tumour cells (DTCs).

STATE OF THE ART

The diagnosis of numerous pathological conditions, in particular that oftumours, is typically performed according to methods characterized, onthe one hand, by different degrees of invasiveness and, on the other, bydifferent levels of performance in terms of specificity, sensitivity,and reliability.

Some methods of screening are based upon analyses aimed at detecting thepresence of biomarkers in the peripheral bloodflow (for example, CA-125,CEA, PSA) or in other biological fluids (for example, test of occultblood in the faeces or test on faecal DNA). Said methods, however, arebased upon an indirect evaluation that is a function of theconcentration of a biomarker and not on the direct analysis, forexample, of tumoural tissue. Consequently, their sensitivity andspecificity are not particularly high.

In the case, considered by way of example, of prostate cancer, one ofthe most widespread forms of cancer in the male population(approximately one quarter of all the cases of cancer, 300,000 new casesa year in Europe), screening on the basis of serological parameters(dosages of PSA) is widespread, but has a poor sensitivity andspecificity.

More invasive procedures, such as digital rectal examination can attimes complete the clinical picture, but more frequently it is necessaryto carry out a prostatic biopsy to have a more accurate diagnosis, in anescalation of invasiveness that results in a growing discomfort for thepatient and in non-negligible costs for the health system.

Similar situations arise in other contexts, such as cancer of thecolon-rectum, where recourse is frequently had to the evaluation ofoccult blood in the faeces, or else an invasive analysis is performedvia colonoscopy and, possibly, a biopsy with sampling of tissues takendirectly from the colon-rectum.

Since, in this context, early diagnosis enables on average a moreeffective treatment, it is obviously of interest to evaluate the onsetof cancer at an early stage and with a high degree of sensitivity andspecificity.

In this sense, recent studies (for example, Zieglschmid et al., Crit.Rev. Clin. Lab. Sci. 42, 155-196 (2005)) on circulating tumour cells(CTCs) have demonstrated that, in the peripheral bloodflow, cellsderiving from the primary tumour are found. Even though they areextremely rare, the possibility of identifying them and isolating themconstitutes a potentially very interesting alternative to the invasivemethods described previously for obtaining specimens of tumoural tissueto be characterized, subsequently, for diagnostic purposes. Regulatorybodies, such as the FDA, have already approved some systems for clinicaluse based upon CTCs for evaluation of patients presenting metastases.

For the majority of patients affected by a cancer, in fact, it is notthe main tumour that causes death in so far as, if identified at asufficiently early stage, it can be eliminated surgically, by means ofradiotherapy, chemotherapy, or by a combination of said methods.Instead, the most frequent cause of death are metastases, i.e., tumouralcolonies originated by malignant cells that detach from the main tumourand migrate towards other districts of the body, even at a considerabledistance from the main tumoural site. Since they are difficult toidentify and eliminate, it is frequently impossible to treat all themetastases successfully. Consequently, from the clinical standpointtheir formation can be considered the conclusive event in the naturalprogress of a cancer.

Consequently, since there has been demonstrated, in the case of breastcancer, cancer of the colon, and cancer of the prostate, a correlationbetween the number of CTCs and the increased risk of unfavourableoutcome of the illness, it appears evident that the possibility ofisolating them and, by means of molecular analysis, obtaining a completeinformative picture therefrom having clinical importance, would havevery significant diagnostic repercussions.

It has moreover been proven that the analysis of disseminated tumourcells (DTCs), i.e., of tumour cells that can be found in the bone marrowor in the lymph nodes, is, in certain contexts, even more significantthan the analysis of the primary tumour. Some research groups, forexample, have noted (Klein et al., Tumour cell. 13(5), 441-53 (2008))that the over-expression of HER2, which can be detected in a single DTCbut not in the primary tumour, proves predictive of a very high risk ofdeath.

Furthermore, it is known, for example from the patent No. EP1109938,that the analysis of single multiple cells can be more informative ascompared to the analysis of a set of cells. In particular, it has beenfound (Klein et. al., Proc. Natnl. Acad. Sci. USA 96, 4494-4499 (1999))that different DTCs of one and the same patient have differentchromosome variations (losses and amplifications in different sites).

At the current state of the art, for isolation of circulating anddisseminated tumour cells there are, however, available approaches of achiefly analytical nature that are very complex and laborious, which,however, have limited yields and generate specimens distinguished by alow degree of purity. Consequently, said specimens of CTCs or DTCs arenot compatible with some of the most refined and reliable diagnosticprocedures, such as sequencing. The limited purity, in fact, determinesa high risk of false positives and false negatives caused by wrong callsin the bases of the sequence.

Attempts have been made (Nagrath et al., Nature, 450, 1235-1241 (2007))to isolate CTCs by means of microfluidic devices with an inner surfacecoated with anti-EpCAM antibodies (expressed in epithelial cells but notin leukocytes) that are able to process volumes of blood of the order ofmilliliters, without having to resort to preliminary treatments thatentail operations with a high risk of loss of part of the cells ofinterest (for example, centrifugation, flushing and incubation).However, said methods have led to levels of purity on average of50%-60%, which do not prove sufficient for carrying out, downstream,analyses for which the purity of the specimen is crucial, such as, forexample, the evaluation of copy-number variations, microsatelliteinstability, loss of heterozygosity (LoH), gene expression in which abetter signal-to-noise (S/N) ratio is fundamental for diagnosticapplications, or identification of new drugs, or again in the sector ofresearch into cancer-initiating cells.

Similar considerations can be extended to other methods the enormousdiagnostic potential of which has been highlighted, which, however,cannot be concretely implemented since the known procedures ofenrichment of biological specimens and isolation of a population of rarecells therein do not enable sufficient levels of purity to be obtained,if not following upon a heavy, complex and costly analytical workloadthat cannot, consequently, find application in medical practice. Forinstance (Bonci et al. “The miR-15a-miR-16-1 cluster controls prostatecancer by targeting multiple oncogenic activities” Oct. 19, 2008;doi:10.1038/nm.1880), it has recently been demonstrated that somemicroRNAs (miRNAs), i.e., short single-strand non-encoding fragments ofRNA, containing approximately 22 nucleotides, are directly involved inthe development and progress of cancer. From a study of the expressionof miR-15a and miR-16 in cells of a primary tumour (prostate cancer) bymeans of quantitative PCR, there has been noted a considerableunder-regulation of both of the miRNAs at compared to the correspondinghealthy tissue. Said datum has been confirmed by means of in-situhybridization (ISH). It has moreover been demonstrated that the deletionof the miR-15a-miR-16 cluster is generally associated to advanced stagesof the illness, even though in some cases the under-regulation of thesemiRNAs has been recorded already during the initial stages of thedevelopment of cancer. Consequently, from the diagnostic standpoint, thepossibility of isolating tumour cells and of evaluating whether theypresent an under-regulation of these miRNAs would provide a preciseindication on the state of advance of the illness, which is useful tothe physician also for the choice of the most appropriate form oftherapy.

To proceed with further considerations of a therapeutic nature, it isworthwhile recalling that some therapeutic approaches in the oncologysector are based upon the use of monoclonal antibodies that are able tohave a direct influence upon the survival of tumour cells, deprivingthem of essential signals of extracellular proliferation, such as thosemediated by growth factors through their cell receptors. For example,one of the targets of interest, in this context, is theepidermal-growth-factor receptor (EGFr). Binding between the epidermalgrowth factor (EGF) and the corresponding receptor EGFr triggers acascade of cellular biochemical events, which includeautophosphorylation and internalization of EGFr, which culminates incell proliferation.

It has been shown that both EGF and the transforming growth factor-α(TGF-α) bind to EGFr and lead to cell proliferation and growth of thetumour. In many cases, the increased expression of EGFr has beenaccompanied by the production of TGF-α or EGF by the tumour cells, thussuggesting involvement of an autocrine growth control in the progress ofcancer. Consequently, there has been proposed the use of antibodiesagainst EGF, TGF-α and EGFr in the treatment of tumours in which theyare expressed or over-expressed. It has recently been demonstrated, asdescribed in the international patent application published under No.WO28112274, the presence of a mutation of which in the K-RAS gene or inthe B-RAF gene in tumour cells constitutes an indication on the factthat the tumour will not respond to the treatment with an agent designedto bind to a polypeptide of EGFr.

It will be understood that the possibility of detecting, starting from aspecimen of tumour cells taken from a patient by means of a non-invasiveprocedure, the presence of such a mutation would provide the physicianwith a tool of enormous importance for evaluating whether to proceedwith a therapy based upon the use of an anti-EGFr monoclonal antibody orelse reject it, knowing beforehand that it would not be effective. Alsoin this perspective, however, the methodologies of enrichment,identification and isolation of rare cells in the blood, or in an somebiological fluid, do not enable a sufficient purity to be obtained toguarantee that the subsequent analysis will be reliable (exclusion offalse positives, false negatives, etc.) and, in some cases, that theycan even materially be conducted (for example, for the cases in which,since the purity is low, the signal-to-noise ratio is too low to obtaina valid reading).

SUMMARY OF THE INVENTION

Consequently, an aim of the present invention is to provide a method foridentification, isolation, and subsequent analysis of circulating ordisseminated rare cells obtained by means of a sampling of anon-invasive nature that will overcome the drawbacks of the known artdescribed previously.

Consequently, provided according to the present invention are proceduresfor diagnostic purposes to be carried out on a specimen of cells ofinterest obtained starting from an organic fluid taken from a patient,which are identified and subsequently isolated until a purity of atleast 90% is obtained, operating according to the method specified inClaim 1.

Preferably, the step of isolating at least one cell from amongst said atleast one type of circulating tumour cells or disseminated tumour cellsor portions thereof is performed so as to obtain a specimen of tumourcells or portions thereof having a purity of at least 95%. Even morepreferably, the step of isolating at least one cell from amongst said atleast one type of circulating tumour cells or disseminated tumour cellsor portions thereof is performed so as to obtain a specimen of tumourcells or portions thereof having a purity of 100%.

In particular, according to a preferential embodiment of the presentinvention the step of isolation of the rare cells is performed accordingto a technology developed by the present applicant and based upon theuse of a silicon microchip that integrates several hundreds of thousandsof electrodes of micrometric dimensions by manipulating individual cellsso as to isolate the cells of interest with a unitary purity renderingthem available for molecular analysis. In this way, the method accordingto the invention provides cells with a high purity, is highly automatedfor the most delicate part of the process (isolation of individualcells), and enables implementation of analytical techniques of highdiagnostic and predictive reliability. In the light of what has beendescribed previously, there will emerge clearly the innovative scope, ata diagnostic and therapeutic level, of the present invention, whichenables improvement of the oncological treatment in the different stagesof the course of the illness, rendering possible both a non-invasiveearly diagnosis having a level of reliability comparable with a biopsy,and a sensitive and accurate follow-up in the course of the treatmentwith specific cancer drugs and/or in the post-operative stage in orderto follow the evolution of the illness and the quality of the responseto the clinical treatments, both in the metastatic stage and in theadjuvant stage.

According to the present invention, an organic fluid taken in anon-invasive manner from a patient is, in a first instance, processed bymaking one or more passages of enrichment of the cells of interestaccording to one or more methodologies known in the art, such as Ficoll,selective lysis of the erythrocytes, filtration with filters based upondimension of the cells—obtained by means of photolithographicmicromachining or other technique, such as track-etched membranes,depletion or magnetic enrichment. The enriched cells are then labelledwith specific antibodies for identifying the cells of interest and/orthe contaminating cells, etc.

In this context, by the expression “organic fluid” or “corporeal fluid”reference is made to a fluid obtained starting from a corporeal specimenin which there is a high probability of finding cells of interest. Theorganic fluid can be obtained from the corporeal specimen, eitherdirectly—such as for example, peripheral bloodflow, bone marrow,urine—or indirectly, such as for example, by means of trypsinization ofa tissue from a lymph node.

In this context, by the expression “cells of interest” reference is madeto cells, the characteristics of which, detectable by means ofappropriate techniques of analysis, can provide indications of adiagnostic or therapeutic nature on a pathological condition of thepatient.

For instance, the corporeal fluid is peripheral blood, which can bedrawn in a substantially non-invasive way from the patient, according tothe methodologies commonly in use, and the cells of interest arecirculating tumour cells (CTCs).

As further example, the organic fluid is blood from bone marrow, and thecells of interest are disseminated tumour cells (DTC).

The method of the invention is then characterized by the use of amicrofluidic system that is able to select individually single cellsfrom the enriched specimen and isolate them in an automatic orsemi-automatic non-manual way.

Used hereinafter for reasons of simplicity is the term “single cells” or“individual cells”, but this term must be understood as including asingle cell or a single aggregate of cells in so far as, in general, itmay occur that the cells of interest present as not isolated but boundto one or more other cells, whether tumour cells or non-tumour cells.Furthermore, there must be understood also the possibility of analysingportions of cell, such as isolated nuclei.

Via said isolation of the cells in the microfluidic system, a set isobtained containing only cells of interest, i.e., a specimen of a purityequal to 100%, which thus proves suitable for being subjected to aplurality of procedures of molecular analysis.

Advantageously, in the aforesaid microfluidic system for the individualselection of single cells, the selection is made on the basis of imagesof the cells themselves.

Advantageously, said images of the cells are acquired in the absence offlow in the medium of suspension of the cells themselves.

Advantageously, said images comprise images acquired in fluorescence.

By “microfluidic device” is understood, herein, a device designed tomanage volumes of liquid in a laminar-flow regime, in which the spacefor containing the liquid during analysis has at least one dimensionsmaller than 1 mm.

By “device capable of selecting individually single cells” is hereunderstood a device that is able to carry out selection of one or moresingle cells, one at a time or simultaneously, on the basis ofparameters evaluated individually on each cell.

By “non-manual isolation” is understood a movement of the cells in whichmanuality of the operator is not required, such as for example, in thecase of movement by means of mobile dielectrophoretic cages.

By “automatic isolation” is understood a movement of the cells in whichintervention of the operator is not required, such as for example, inthe case of movement by means of dielectrophoretic cages managed by aprogram executed in a substantially non-interactive way by amicroprocessor.

By “semi-automatic isolation” is understood a movement of the cells inwhich the interaction with the operator enables exerting an indirectcontrol on the manipulation, such as for example, in the case ofmovement, for example by means of dielectrophoretic cages, managed by aninteractive program executed by a microprocessor.

Advantageously, the aforesaid selection is made in an automatic orsemi-automatic way.

By “automatic selection” is understood herein a selection of the cellsin which intervention of judgement of the operator is not required, suchas for example, in the case where there is determined automatically withan automatic classifier which cells correspond effectively to tumourcells of interest. For example, this could be performed with aclassifier of images that processes the acquired images of the cells,extracts the discriminating features therefrom and classifies them onthe basis of an algorithm.

By “semi-automatic selection” is understood herein a selection of thecells in which intervention of judgement of the operator is required.For instance, this could be performed with a system that acquires theimages of the cells, extracts the discriminating features therefrom, andproposes them to the operator for the final decision as to whether theyshould be selected.

The molecular analysis on the cells recovered can then be executed viadifferent techniques—thanks to the high purity of the specimenrecovered—, amongst which, by way of non-limiting example:

-   -   sequencing (for example, for identification of mutations);    -   microsatellite analysis (for example, with Quantitative        Fluorescent PCR, i.e., QF-PCR);    -   comparative genomic hybridization (CGH);    -   array CGH;    -   end-point PCR;    -   real-time PCR;    -   methylation analysis:        -   quantitative methylation analysis with pyrosequencing;        -   bisulphite-genomic-sequencing PCR (BSP);        -   methylation-specific PCR (MSP);        -   Combined Bisulphite Restriction Analysis of DNA (COBRA)        -   methylation-sensitive single-nucleotide primer extension            (MS-SNuPE)    -   analysis of gene expression;        -   RT-PCR;        -   single-cell gene expression;        -   digital PCR.

Further characteristics and advantages of the invention will emergeclearly from the ensuing description of some non-limiting examples ofembodiment thereof, provided with reference to the figures of theannexed drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a flowchart summarizing an embodiment of the non-invasivemethod of diagnosis according to the present invention;

FIG. 2 is a schematic illustration of an example of device forimplementing the method (or the substantial and characterizing partthereof) according to the invention;

FIGS. 3A, 3B and 3D show images acquired in the course of scanning ofcirculating tumour cells according to the method of the invention;

FIGS. 4A, 4B, 4C and 4D show a series of electropherograms obtained inthe course of the genetic analysis of circulating tumour cellsidentified and isolated according to the method of the invention;

FIG. 5 shows images acquired in the course of scanning of circulatingtumour cells selected and isolated according to the method of theinvention from peripheral bloodflow of patients affected by mCRC;

FIGS. 6 and 7 show, respectively, spectra regarding the detection of amutation Gly2Val on K-RAS (respectively SEQ ID NO:1 and 2) of CTCsisolated from the peripheral bloodflow of a patient affected by mCRCaccording to the method of the invention;

FIGS. 8, 9 and 10 each show five images acquired in the course ofscanning of an enriched specimen, said images regarding the contents offive dielectrophoretic cages on a DEPArray™ chip. The cells of eachfigure were recovered together with one another and separately from thecells of the other figures.

FIG. 10 shows circulating tumour cells according to the method of theinvention and FIGS. 8 and 9 the corresponding negative controls (normalleukocytes).

DETAILED DESCRIPTION

The subject of the present invention is a method of identification,isolation, and subsequent analysis of rare cells, in particularcirculating tumour cells or disseminated tumour cells, obtainedpreferably by means of a sampling of a non-invasive nature.

Sampling

The specimens can be taken from the peripheral circulation of thepatient, or else from bone marrow through various techniques known inthe sector.

Pre-Enrichment

The proportion of tumour cells in the specimen taken can be enrichedusing various methods, such as for example: centrifugation on densitygradient, constituted by solutions such as Ficoll or Percoll; mechanicalenrichment, such as filters of various types; enrichment by means ofseparation by dielectrophoresis via a purposely provideddevice—dielectrophoretic-activated cell sorter (DACS); selective lysis,such as for example, selective lysis of erythrocytes not of interest;immunomagnetic separation, via immunomagnetic beads with positiveselection (using beads bound to specific antibodies for the populationto be recovered) or with negative selection (depletion of cellpopulations that are not of interest), and in which the two types ofselection can be coupled in order to increase the specificity of theprocedure; FACS, on cells labelled with a specific fluorescent antibody;solid-phase immunoseparation, advantageously by means of microfluidicsystems presenting surfaces coated with specific antibodies forepithelial receptors (such as EpCAMs).

For the majority, these procedures can be automated and all the sortingprocedures can be preceded by separation by centrifugation on densitygradient or alternatively they can be applied on whole blood.

In general, the process starts with a dilution, but this is not strictlynecessary for all techniques.

Other Enrichment Techniques

A further technique well known to persons skilled in the branch is theone referred to as MACS developed by Miltenyi Biotech, or else Easy-sep,developed by Stem-cell technologies.

Alternatively, it is possible to use paramagnetic beads of largerdimensions that do not require the use of a special column, but can alsobe used when working with wells or test tubes (such as, for example,anti-EpCAM Dynabeads).

To sum up, the step of enriching the specimen in the population of cellscomprising at least one type of tumour cells can be performed via amethod consisting of successive steps by means of a selection of cellsmade on the basis of at least one parameter chosen from the groupconsisting of:

a. mass density;

b. morphology;

c. electrical properties;

d. chemical properties;

e. mechanical properties;

f. expression of surface antigens;

g. expression of intracytosolic antigens;

h. dielectric properties;

i. magnetic properties;

l. geometrical properties (size, etc.); and

m. optical properties.

or combinations thereof.

Advantageously, the enrichment of the tumour cells is then furtherobtained by means of a second step, in which the positive or negativeselection of cells is made from the mononucleated cells recovered in thefirst step. Obviously, the second enrichment step can comprise aselection made on the basis of the capacity to express or not a specificantigen, evaluated with one of the following techniques:

a. MACS, i.e., Magnetic-Activated Cell Sorter;

b. DACS, i.e., Dielectrophoretic-Activated Cell Sorter;

c. FACS, i.e., Fluorescence-Activated Cell Sorter.

Isolation of Single Tumour Cells

Next, the specimen containing the tumour cells is inserted in amicrofluidic device that is able to select individually single cells andisolate them in an automatic or semi-automatic non-manual way. For thispurpose, it is possible to use a dielectrophoretic isolation (DEPArray,using for example, the techniques described in PCT/IB2007/000963 or inPCT/IB2007/000751, or again in Manaresi et al., IEEE J. Solid-StateCircuits, 38, 2297-2305 (2003) and in Romani et al., Proceedings of theInternational Solid State Circuit Conference, 1, 224-225 (2004)), orelse opto-electronic traps or optophoretic isolation or again, lasertweezers (see, for example, Reichle et al., Electrophoresis, 22, 272-82(2002) or Fiedler et al., Anal. Chem., 70, 1909-15 (1998)).

The contents of said documents are incorporated herein as regards therelevant parts just for reference.

Identification of the cells of interest can be made, for example, viasensors:

-   -   external sensors;    -   optical sensors, such as a fluorescence microscope;

or also

-   -   internal sensors;    -   optical sensors, as disclosed in the patents Nos. WO2007049103        and WO2007010367, which describe an integrated method of        identification of the fluorescent cells;    -   impedentiometric sensors, as disclosed in the patents Nos.        WO2007049103 and WO2007010367, for the detection of        impedentiometric characteristics of cells.

The signal for selection of the cells can be also indirect, such as, forexample, the presence of a microbead coupled to an antibody, which is inturn coupled to the cell. The presence of one or more microbeads coupledto a cell can be detected as already described previously, by means ofimpedentiometric sensors or optical sensors (in clear field or influorescence).

According to the invention, by manipulating individually single tumourcells by means of a microfluidic device, preferably in an automated way,the tumour cells contained in the pre-enriched specimen taken areisolated to constitute a specimen to be analysed having a purity of atleast 90%.

Preferably, tumour cells are isolated to constitute a specimen to beanalysed having a purity higher than 95%. Even more preferably, tumourcells are isolated to constitute a specimen to be analysed having apurity of 100%.

Genetic Analysis

On the tumour cells recovered according to the invention various typesof analysis can be performed that enable genetic or chromosomalcharacterization thereof at different levels of resolution andsensitivity and according to the diagnostic purpose of the study, inaccordance with what has been described previously.

For example, it is possible to proceed to sequencing of CTCs taken frompatients presenting metastases in order to detect the presence ofmutations of the K-RAS gene.

In the case of prostate cancer, it is possible to evaluate the presenceof deletions on CTCs by means of single-cell whole-genome amplification.

Again, in the case of cancers of unknown primary origin (CUPs) it ispossible to identify the original tissue via the genetic profile and/orthe profile of expression of the CTCs, thus obtaining information thatis fundamental for the choice of the most appropriate therapy.

In the case of disseminated tumour cells, then, it is possible toperform analysis of gene expression on single CTCs and/or DTCs in orderto identify prognostic indicators and potential therapeutic targets.

PREFERRED EXAMPLES OF EMBODIMENTS OF THE INVENTION

By way of non-limiting example, described in what follows is apreferential embodiment of the method according to the presentinvention, following the flowchart represented in FIG. 1.

Starting Specimen

In a specimen of 17 ml of whole blood taken from a healthy male donorthere were introduced by spiking 2000 cells belonging to the cell lineof breast cancer MCF7.

Preliminary Enrichment

The preferential embodiment of the invention envisages a processconsisting of successive steps.

(a) FICOLL.

The specimen, was treated with an anticoagulant (e.g., EDTA) andtransferred, preferably within 8 h, into a 50-ml sterile test-tube madeof polypropylene. From this a volume of 50 μl was extracted, which wasused for counting white blood cells (WBCs) and red blood cells (RBCs)using a Coulter counter.

The specimen was then diluted 1:4 with PBS and EDTA (2 mM) having a pHof 7.2.

A volume (≤30 ml) of diluted blood was accurately stratified on 15 ml ofFicoll-Hypaque (density=1.077 g/ml).

Next, the specimen was centrifuged at 654 g for 30 min at 22° C. in a(brakeless) tilt-rotor centrifuge.

The plasma was removed with a pipette (of the Pasteur type)—but anautomatic pipettator may also be used—taking care not to interfere withthe layer of lymphocytes at the interface. The layer was carefullycollected with a drop-counter and transferred into a 50-ml polypropylenesterile conical test-tube.

The test-tube was filled with PBS and EDTA (2 mM), shaken andcentrifuged at 300 g for 10 min at 10° C.

The supernatant portion was rejected.

The test-tube was filled with PBS and EDTA (2 mM), and again shaken andcentrifuged at 300 g for 10 min at 10° C. The supernatant portion wasdrawn off.

From the specimen the cell pellet (i.e., the complex of theperipheral-bloodflow mononucleated cells, PBMNC) was taken, to bere-suspended in 5 ml of buffer. From this was taken an aliquot of 50 μlto carry out the WBC count with a Coulter.

The test-tube was filled with buffer, shaken and centrifuged at 200 gfor 10 min at 10° C. The supernatant portion was rejected.

An alternative to Ficoll as first step of pre-enrichment for eliminatingthe erythrocytes is represented by selective lysis of erythrocytes,which exploits chemical properties of the cells.

(b) CD45 MACS Depletion

The cell pellet was re-suspended in 80 μl of buffer for a total of 10⁷cells.

An amount of 20 μl of microbeads of anti-CD45 were added for a total of10⁷ cells. There followed steps of shaking and incubation for 15 min at4° C.

The cells were washed by filling the test-tube with buffer andcentrifuging at 300 g for 10 min at 10° C.

The supernatant portion was rejected and the cell pellet wasre-suspended in 500 μl of buffer for a total of 10⁸ cells.

The MACS column of LS type (Miltenyi) was positioned within the magneticfield of a suitable MACS separator, according to the indicationsprovided by the manufacturer.

A pre-filter was positioned on each column.

Both the pre-filter and the column were prepared by rinsing with 3 ml ofbuffer.

The cell suspension was applied to the column (or on the pre-filter).

The test-tube that contained the cell suspension, now empty, was filledwith 9 ml of buffer.

The non-labelled cells were collected, and the column was washed byadding for three times 3 ml of buffer, taking it each time from thetest-tube filled in the previous step.

The column was removed from the separator and set on a test-tube.

An amount of 5 ml of buffer were introduced into the column using apipette.

The fraction containing the magnetically labelled cells was expelledimmediately afterwards by operating the plunger.

To reduce the number of contaminating cells further, it is possible touse a depletion cocktail with further magnetic microbeads functionalizedwith antibodies that recognize antigens present selectively on cells tobe depleted, such as for example, anti-GPA microbeads (to eliminate theresidual erythrocytes from the Ficoll).

To reduce the number of contaminating cells even further, it is possibleto carry out a second passage in a magnetic column.

(c) Fixing and Labelling

The post-MACS cells were centrifuged at 300 g for 10 min; the pellet wasre-suspended in 40 μl of buffer.

The specimen was transferred into a 1.5-ml test-tube. To this were added760 μl of 4% paraformaldehyde just prepared, followed by incubation for20 min at room temperature.

After centrifugation at 0.2 r.c.f. r.p.m. for 5 min in themicrocentrifuge, the supernatant fraction was drawn off.

This was followed by washing with 1 ml of PBS, centrifugation at 0.2r.c.f. r.p.m. for 5 min in the microcentrifuge, and the supernatantfraction was again aspirated.

To this were added 100 μl of PBS/BSA at 3% (blocking buffer).

The specimen was incubated for 10 min at room temperature.

After further centrifugation at 0.2 r.c.f. r.p.m. for 5 min in themicrocentrifuge, followed by aspiration of the supernatant fraction,staining was performed with EpCAM-FITC and CD45-PE (Miltenyi, accordingto the protocol recommended by the manufacturer).

To complete the process, a final washing was performed with 1000 μl ofSuper Buffer (Hepes 400 mM, 1% BSA) and 1 μl of Hoechst 33324 dilutedwith water (100 μg/ml) and the specimen was vortexed.

The specimen was subjected to a quality control to verify the intensityof fluorescence of the marker and the total cell concentration. A partof the labelled specimen was re-suspended in a minimum volume ofspecific buffer, useful for dielectrophoretic manipulation, and wasloaded on a device for control of quality of the specimen and controlledunder the fluorescence microscope: the intensity of fluorescence of thecells was noted in the different channels and a count was made of thecells labelled in Hoechst 33324 (total nucleated cells). If the cellconcentration was higher than the optimal one for proper operation ofthe device for isolation of the single cells, the next step was dilutionof the specimen to obtain the desired concentration.

Isolation

The cells thus obtained were then inserted in the chip DEPArray®CONV600K (manufactured by Silicon BioSystems, see for example, thedocument No. WO0069525) for dielectrophoretic manipulation and isolationof the tumour cells. The device as a whole is illustrated in FIG. 2.

The specimen was subjected to scanning, identification, and selection,sorting, and recovery of the tumour cells.

The caged cells were scanned in an automatic or manual way at themicroscope with 3 different fluorescence channels (i.e., in 3 differentwavelengths). Observation in the DAPI channel (where by “DAPI channel”is meant UV excitation and emission in the blue, which is hence usedalso for visualization of Hoechst 33324) enables identification of the(positive) nucleated cells, whilst observation in the channels for EpCAMand CD45 enables differentiation between tumour cells (DAPI+, EpCAM+ andCD45−, with compatible morphology), lymphocytes (DAPI+, EpCAM−, CD45+)and spurious signals (DAPI+, EpCAM+ and CD45+ or also DAPI−, EpCAM+ andCD45+, or DAPI+, EpCAM+ and CD45− with morphology incompatible withtumour cells).

FIGS. 3A-B-C show images acquired in the course of scanning of the cellsof the specimen within the DEPArray® CONV600K. More in particular, FIG.3A shows the images for three tumour cells in the three channels takeninto consideration. FIG. 3B shows the images for a spurious cell in thethree channels taken into consideration. FIG. 3C shows the images fortwo lymphocytes in the three channels taken into consideration.

Selection of cells was then performed by selecting the cages containingthe cells found positive to DAPI (nucleated cells), positive to EpCAMand negative, to CD45. Sixteen cells were identified on the chip, someof which were double. Fifteen cells, some of which were double, werefinally recovered in a few microliters (<40 μl) in a 0.2-ml PCR tube.

The final analysis was performed using an Applied Biosystems MinifilerKit.

Given in FIGS. 4A-B-C-D are the results of the analysis of the fragments(QF-PCR) performed using a Minifiler Kit. In the four fluorescencechannels taken into consideration, different microsatellites wereanalysed. It may be noted how in no case were there traces ofcontamination by alleles coming from the male subject into whose bloodthe tumour cells were introduced by spiking.

As an alternative to the anti-EpCAM antibody, it is possible to use adifferent type of antibody, such as, for example, an antibody thatrecognizes one or more types of cytokeratin, which is not expressed inthe blood cells.

Alternatively, it is possible to use an antibody that is morecharacterizing for cancer (tumour-specific), such as for example:

-   -   for the prostate, the Prostate Specific Antigen (PSA);    -   for the lung, the Thyroid Transcription Factor 1 (TTF-1);    -   for the breast, the Human Epidermal growth factor Receptor 2        (HER2/neu)

Possibilities of Application

Obtaining a specimen of CTC having a purity of 100%, following upon anautomatable procedure of selection and isolation, renders feasiblediagnostic pathways for a plurality of conditions that, otherwise, couldnot be analysed accurately, reliably, and precisely.

Example 1

Non-invasive evaluation of mutations (for example, in the K-RAS gene) incancer patients.

Described in what follows is the case of isolation of CTCs fromperipheral bloodflow of patients affected by metastatic Colon-RectalCancer (mCRC).

A specimen of 7.5 ml of peripheral bloodflow was drawn from the patientin Vacutainer tubes with EDTA anticoagulant (Beckton Dickinson). Thespecimen was analysed with Coulter Counter (Beckman Coulter) andpresented 42×10⁶ leukocytes (WBCs) and 43.95×10⁹ erythrocytes (RBCs).The PBMCs were then isolated via centrifugation (in Ficoll 1077). ThePBMCs recovered (16.5×10⁶ on the basis of the count with CoulterCounter) were washed in PBS with BSA and EDTA (Running Buffer, Miltenyi)and were selected via depletion with magnetic microbeads conjugated toanti-CD45 and anti-GPA antibodies (Miltenyi) according to theinstructions of the manufacturer. The resulting cells (negative fractionfor CD45 and GPA) were fixated with 2% PFA in PBS for 20 min at roomtemperature (RT). This was followed by washing in PBS and incubation in3% PBS/BSA (blocking buffer), for 10 min at room temperature. After aflushing in PBS, CD45 labelling was performed with 10 μl ofPE-conjugated anti-CD45 antibody (Miltenyi) in 100 μl of MiltenyiRunning Buffer (RB) for 10 min at 4° C. The reaction was blocked byadding 1 ml of RB, centrifuging and drawing off the supernatant portion.The cells were then permeabilized with 90 μl InsidePerm (Miltenyi) andsimultaneously labelled with 10 μl of FITC-conjugated anti-CK antibodyfor 10 min at room temperature. The reaction was terminated by adding 1ml of Inside Perm, centrifuging, and removing the supernatant fraction.The pellet was re-suspended in buffer optimized for manipulation ofcells fixated and permeabilized with dielectrophoresis—Hepes (400mM)+BSA (2%) in water (SB)—and DAPI (1 mg/ml) and, finally, washed in SBand re-suspended for injection into the chip DEPArray™ CONV600K (with100,000 dielectrophoretic cages). Present on the chip were approximately16,000 cells. After scanning in DAPI/FITC/PE the CTCs were identified.FIG. 5 contains images of CTCs (indicated by a white arrow) selected andisolated by means of the method of the invention, said images beingappropriately processed so as to highlight the fluorescence in threerespective channels: FITC-conjugated anti-CK MoAb (Miltenyi) in thegreen channel; PE-conjugated anti-CD45 MoAb (Miltenyi) in the redchannel; and DAPI labelling of DNA for identification of the nuclei inthe blue channel.

It should be noted how it is possible to distinguish clearly betweenvarious types of possible events from an analysis of the processedimages, whereas in a less refined analysis that is not based uponimages, but only upon the intensity of the overall fluorescence, theimages could be rejected as false positives.

For example, the cage with ID 382 contains a pair of CTCs. These wereevidently already bound at the start, since the probability of two ofthe three CTCs present ending up in one and the same cage is negligible.Without image analysis, this event could have been rejected since, ingeneral, clusters of cells can give rise to spurious fluorescencesignals given that the antibody—for example, anti-CK—gets trapped in anon-specific way. Recovery of the two ID382 cells hence has a purity of100% (and is compatible with the majority of molecular analyses).

The cage with ID 11439 indicates a DAPI+/CK+/CD45+ event. Without imageanalysis said event could have been rejected as spurious, but, instead,since the image makes possible detection of the distribution offluorescence in the event, it is possible to interpret the datum asreferring to a DAPI+/CK+/CD45− CTC (indicated by a white arrow) cagedtogether with three other CK− nucleated cells, two of which CD45+. Thecontents of said cage can be recovered separately. Using a system basedupon mobile dielectrophoretic cages, the segregation in single cages ofthe cells that initially share the same cage can be performed byapplying appropriate patterns of cages. If it is not possible tosegregate the CTC from the other contaminating cells of the cage, it ispossible in any case to recover the contents of the cage, associating tosaid recovery the information of the presence of contaminants. This is afurther advantage of the technique according to the invention. Thepurity of the cells recovered is noted down and this can be taken intoaccount in the analysis downstream. In the case in point, the cell couldhence be recovered together with the other three (a purity of 25%), orelse separated and recovered alone to obtain a pure recovery of 100% (ifisolated alone), or else of 50% if isolated together with a contaminant,or of 90% if isolated together with a contaminant and a further nineCTCs, without contaminants possibly present in other cages (not in thiscase).

The cage ID 5007 shows an event which, on the basis of the totalfluorescence, would appear similar to that of ID 11439 since it is aDAPI+/CK+/CD45+ event. However, on the basis of the image, it ispossible to determine how it is a spurious event in so far as it islinked to a cluster that has trapped anti-CK antibodies.

The cage ID 13103 shows an event that, on the basis of the totalfluorescence, would appear similar to that of ID 11439 since it is aDAPI+/CK+/CD45+ event. However, on the basis of the image, it ispossible to determine how as it is a spurious event in so far as it islinked to a single positive double cell with CD45+ (non-CTC) signal.

The cages with ID 8614 shows an event CK+/CD45− but DAPI−, notclassified as CTC. The cage with ID 7796 shows a control event (twowhite blood cells) comprising two cells that are not classified as CTCs,because they are CK− and one is also CD45+.

The cells of interest can hence be isolated or otherwise, according tothe purposes of the experiment, in terms of purity of the specimen andaccording to the ease of separation of the cell from an agglomerate ofcells within which it is comprised. In this sense, the selection basedupon images is particularly effective for exclusion of false positivesand false negatives.

As an alternative to Ficoll, it is possible to use a technique basedupon selective lysis of red blood cells (RBCs). In this case, in thespecimen there remain in the first instance also the granulocytes. Aftersaid removal of the RBCs, the tumour cells can be further enriched viapositive immuno-magnetic selection (e.g., with magnetic microbeadscoupled to anti-EpCAM antibodies), or negative immuno-magnetic selection(e.g., via magnetic microbeads coupled to anti-CD45 antibodies or to acocktail with anti-CD45 and other antibodies against antigens notpresent in the CTCs). In any case, the few CTCs present are obtained ina specimen containing tens of thousands or hundreds of thousands ofleukocytes.

Isolation of the CTCs from the peripheral bloodflow is likewisecompatible with procedures of enrichment and labelling approved by theFDA, such as the Veridex CellSearch™ system. In this case, theCellSearch system can be used also for carrying out labelling andenrichment in an automated way (with the AutoPrep machine). With thissystem, the few CTCs present are obtained (with an optimal yield ofaround 90%) in a specimen with typically only a few thousands ofcontaminating leukocytes (typical values of 1000-5000). The CellSearchAutoPrep represents an excellent enrichment system, and has as its mainlimitation the fact that it is based upon an EpCAM-positiveimmuno-magnetic selection. Consequently, in some types of tumours, wherethe CTCs do not over-express EpCAM, it would be possible to find only afew CTCs. In such cases, the negative selection is more indicated fordepletion of CD45+ cells.

Detection of the mutational status of the K-RAS gene has considerableclinical importance, since it has been demonstrated that some mutationsof K-RAS are connected to the inefficacy of therapies based uponmonoclonal antibodies directed against the EGFr receptor. Saidmutations, in fact, activate, downstream, the mechanism of cellproliferation in spite of the EGFr inhibition upstream. Therapies withCetuximab and Panitumumab are already indicated only for patients withwild-type K-RAS. The possibility of identifying the mutation in CTCsaffords the possibility of not having to resort to a biopsy, inparticular in all those circumstances in which a biopsy is not possibleor is very complex to perform. Even when tissues removed surgically areavailable, the possibility of analysing CTCs is in any case interesting.The tumours are intrinsically unstable from the genetic standpoint, andthe metastases can derive from cells located in sites distinct from thatof the primary tumour; consequently, the CTCs can reflect better themolecular profile of the cells undergoing metastasis.

In order to verify the possibility of detecting mutations of K-RAS fromCTCs, CTCs from peripheral bloodflow of patients affected by metastaticcancer of the colon-rectum (mCRC) were analysed. Following theenrichment procedure described above, CTCs were identified and isolatedon the DEPArray™. The purity of 100% enables detection of the possiblepresence of the mutation via sequencing: after prior amplification ofthe starting copies, for example with nested PCR, sequencing is carriedout using known techniques.

In the case in point, the analysis downstream was performed using theDOP-PCR technique with the Omniplex kit produced by Sigma. Afteramplification, the product was analysed with a capillary-electrophoresissequencer manufactured by Applied Biosystems.

FIG. 6 shows an example (SEQ ID NO:1) of detection of a mutation in oneof the specimens analysed, which corresponds to a mutation from glycineto valine in the codon 12 (shown in FIG. 7 is the corresponding negativecontrols (SEQ ID NO:2)).

Example 2

Evaluation on CTCs of deletions (for example, in prostate cancer) viasingle-cell whole-genome amplification and CGH array. In this case,there is advantageously performed isolation of the single cells to berecovered in different wells. In this way, the analysis takes intoaccount the heterogeneity of the population, and supplies not only theaverage, but multiple signals regarding each cell so that it is easierto identify mutations present only in some of the tumour cells.

Example 3

Non-invasive identification of the original tissue in CUP tumours, viagenetic profile and/or profile of expression of the CTCs.

Example 4

Analysis of gene expression on single disseminated tumour cells (DTCs)for identification of prognostic indicators, and potential therapeutictargets. Also in this case, there is advantageously performed isolationof the single cells to be recovered in different wells. In this way, theanalysis takes into account the heterogeneity of the population, andsupplies not only the average, but multiple signals for each cell sothat it is easier to identify mutations present only in some of thetumour cells.

Example 5

As already mentioned, in general, what has been said above regarding“isolation of cells” is to be understood as applying also to “isolationof portions of cell”, such as, for example, the nucleus. In fact, thisenables in any case, for certain types of biomolecular information,significant information to be obtained (for example, by evaluating thegenomic DNA contained in the nucleus).

In the example of isolation of a nucleus, the selection of the cells canbe performed via techniques that mark in a distinguishable way thenuclei of tumour cells (CTCs or DTCs) from non-tumour cells, such as forexample, FISH. In this case, the presence of multiple signals revealsthe presence of duplications of genomic DNA (a characteristic of thetumour cells in general not present in normal cells).

In particular, in the example of breast cancer, it is of interest toevaluate duplication of the genomic region regarding the HER2 gene,normally located on chromosome 17. Hence, two types of probes areapplied, one for the centromere of chromosome 17, and one for the HER2gene. If the gene/chromosome ratio is higher than two the cell isconventionally considered positive to the test. Said information has,for example, implications in the use of drugs such as Trastuzumab(Herceptin) that present efficacy only in the case of over-expression ofthe corresponding HER2 receptor. Not only counting, but also identifyingand isolating said cells in a purified form enables acquisition offurther information on the genetic characteristics of the tumour.

Example 6

The presence of copy-number variations (CNVs) in the DNA constitutes atypical characteristic of a tumour. For evaluation of new drugs it is ofconsiderable interest to be able to evaluate the CNVs in order toassociate them to the course of the illness and, potentially, to areasof the genome in which the CNVs have a significant influence on thecourse of the illness (both in the case of favourable prognosis and inthe case of unfavourable prognosis). This information can be helpful inidentifying genes that could be targets for the pharmacological action.Furthermore, in the stage of diagnosis of cancer it may then be possibleto identify in a minimally invasive way the CNV profile of the CTCs ofthe patient in order to orient the therapy on the basis of theexperience acquired.

To demonstrate the possibility of determining CNVs small specimens ofCTCs and, accordingly, small negative-control specimens were separatedand isolated.

From a patient suffering from metastatic breast cancer (mBrCa), aspecimen of 7.5 ml of peripheral blood was taken and put in a CellSavetube (Veridex). The specimen was enriched and labelled with fluorescentantibodies (PE-conjugated anti-CK, APC-conjugated anti-CD45) and DAPI,with the CellSearch AutoPrep, according to the standard procedure. Theenriched cells were extracted from the Veridex cartridge, andre-suspended in a reduced volume. Said specimen was injected on the chipDEPArray™ A300K (Silicon Biosystems S.p.A.)—with 307,200 electrodes anda number of programmable cages, typically between 19,200 and 76,800.After scanning, there were isolated two negative controls with fivecells, two specimens with five CTCs, one specimen with a single CTC.

FIGS. 8 and 9 illustrate the cells recovered, respectively, in a firstand second recovery with five leukocytes each (DAPI+/CD45+/CK−) asnegative control for the CNV detection. FIG. 10 illustrates the fiveCTCs recovered separately, which form the subject of CNV analysis.

After selection and isolation performed using the method according tothe invention, and given that a specimen is now available with a degreeof purity higher than 90% (in the case in point, 100% for the recoveryof CTCs illustrated in FIG. 10), it is possible to carry outsuccessfully analysis based upon whole-genome amplification, accordingto a methodology illustrated in the document No. EP1109938,ligation-mediated PCR, followed, by metaphase comparative genomichybridization (CGH) or CGH array, which, with specimens of lower purity,would be impossible or intrinsically unreliable in so far as the signaldetected would be too weak or fuzzy in the presence of non-tumouralcells.

The invention claimed is:
 1. A method comprising the steps of: a)enriching a specimen of biological fluid in cells of interest selectedfrom the group consisting of circulating tumour cells and disseminatedtumour cells, wherein the organic fluid is obtained from a subject andcomprises the cells of interest and other cells, wherein the enrichedsample comprises the cells of interest, contaminating cells, and asuspension medium; b) isolating cells of interest from the enrichedsample by: labelling the sample with: an antibody that binds to thecells of interest, the antibody being conjugated with a firstfluorescent marker exciting at a first wavelength, wherein the antibodyis an anti-cytokeratin antibody or an anti-EpCAM antibody, an anti-CD45antibody that binds to the contaminating cells and does not bind to thecells of interest, the second antibody being conjugated with a secondfluorescent marker that excites at a second wavelength, and a thirdfluorescent marker that binds to nucleated cells and excites at a thirdwavelength, wherein the cells of interest in the labelled samplegenerate a distinct combination of three signals when scanned with threedifferent fluorescence channels at first, second, and third wavelengths,wherein the distinct combination of three signals is positive to thefirst fluorescent marker, negative to the second fluorescent marker, andpositive to the third fluorescent marker; introducing the labelledsample into a single closed chamber of a microfluidic device forcontaining the sample, wherein the single closed chamber contains theentire labelled sample and the single closed chamber is bounded by a topsurface adjacent to at least one electrode and a bottom surface that isflat and adjacent to an array of electrodes; applying to the array ofelectrodes a first pattern of voltages to generate a first field offorce acting on the cells of interest and the contaminating cells toindividually trap the cells of interest and the contaminating cells inan array of dielectrophoretic cages; scanning the labelled sample loadedinto the single chamber of the microfluidic device with three differentfluorescence channels at first, second, and third wavelengths, whereinthe sample is scanned in the absence of flow of the suspension medium ofthe labelled sample in the chamber; individually selecting single cellsof interest in the labelled sample that have the distinct combination ofthree signals from the three different fluorescence channelscorresponding to the labelled cells of interest; and individuallymanipulating the selected cells of interest into a purified specimen byapplying to the array of electrodes a second pattern of voltages togenerate a second field of force acting only on the selected cells ofinterest to shift the cells of interest into the purified specimen or byapplying an optical force acting only on the selected cells of interestto shift the selected cells of interest from the dielectrophoretic cagesto the purified specimen, wherein the purified specimen has a purity ofat least 90% as defined by the ratio of the number of the cells ofinterest to the total number of cells in the purified specimen, whereinthe purified specimen has a purity sufficient to allow a molecularanalysis to be performed to detect at least one characteristic of saidcells of interest enabling a diagnosis.
 2. The method according to claim1, wherein the purified specimen has a purity of at least 95%.
 3. Themethod according to claim 2, wherein the purified specimen has a purityof 100%.
 4. The method according claim 1, wherein said individualselection of said cells of interest is made on the basis of theevaluation of fluorescence images of said cells.
 5. The method accordingto claim 1, wherein said step of enriching said specimen of saidbiological fluid comprises the step of treating said specimen of saidbiological fluid so as to separate and recover nucleated cells andenrich said specimen subsequently in nucleated cells.
 6. The methodaccording to claim 5, wherein said step of enriching said specimen ofsaid biological fluid comprises at least the step of centrifuging saidspecimen of said biological fluid in density gradient.
 7. The methodaccording to claim 5, wherein said step of enriching said specimen ofsaid biological fluid comprises at least the step of carrying out aselective lysis of erythrocytes.
 8. The method according to claim 1,wherein said microfluidic device is used equipped with a plurality ofdifferent chambers, separated from one another and hydraulicallyconnected, delimited on at least one face by a single chip or by aplurality of separate chips.
 9. The method according to claim 1, whereinmolecular analysis is performed by means of a procedure selected fromamong: sequencing microsatellite analysis comparative genomichybridization (CGH) array CGH end-point PCR real-time PCR methylationanalysis: quantitative methylation analysis with pyrosequencingbisulphite-genomic-sequencing PCR (BSP) methylation-specific PCR (MSP)Combined Bisulphite Restriction Analysis of DNA (COBRA)methylation-sensitive single nucleotide primer extension (MS-SNuPE)gene-expression analysis RT-PCR single-cell gene expression digital PCR.10. The method according to claim 9, wherein said molecular analysiscomprises assessing the presence of point mutations.
 11. The methodaccording to claim 10, wherein said point single-nucleotide mutationsare in the K-RAS and/or B-RAF gene and the presence of said pointmutations is an indication that the patient will not respond to apredetermined therapy.
 12. The method according to claim 1, wherein saidmolecular analysis comprises evaluating the presence of hypermethylationof oncosuppressor genes.
 13. The method according to claim 1, whereinsaid molecular analysis comprises evaluating the presence of deletionsand/or duplications.
 14. The method according to claim 13, wherein saidmolecular analysis comprises evaluating the presence of deletions in thechromosome region 13q14 in tumour cells in patients with suspectedprostate cancer.
 15. The method according to claim 1, wherein saidmolecular analysis comprises evaluating the over-expression of at leastone of the genes BCL2, CCND1 and WNT3A in tumour cells in patients withsuspected prostate cancer.
 16. The method of claim 1, wherein theCytokeratin is selected from the group consisting of Cytokeratin 8,Cytokeratin 18, Cytokeratin 19, and Cytokeratin
 20. 17. The method ofclaim 1, wherein the selected cells are of interest are manipulated byapplying to the array of electrodes the second pattern of voltages togenerate the second field of force acting only on the selected cells ofinterest to shift the selected cells of interest from thedielectrophoretic cages into the purified specimen.
 18. The method ofclaim 1, wherein the microfluidic device is a DEPArray chip.